Recovery and characterization of polyhydroxyalkanoates produced by recombinant escherichia coli

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Recovery and characterization of polyhydroxyalkanoates produced by recombinant escherichia coli

 

Author: Leung, Choi-fong Joyce
Title: Recovery and characterization of polyhydroxyalkanoates produced by recombinant escherichia coli
Degree: M.Sc.
Year: 2002
Subject: Hong Kong Polytechnic University -- Dissertations
Poly-beta-hydroxyalkanoates
Department: Multi-disciplinary Studies
Dept. of Applied Biology and Chemical Technology
Pages: viii, 83 leaves : ill. ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1615445
URI: http://theses.lib.polyu.edu.hk/handle/200/349
Abstract: A recombinant strain of Escherichia coli XL1-Blue harboring pKS plasmid was cultured for polyhydroxyalkanoate (PHA) production. Cosubstrate of glucose and propionate was used to enhance the incorporation of hydroxyvalerate units for production of PHB-PHV copolymer. J1 medium, with higher concentrations of glucose and propionate at 320 g and 64 g per liter, was fed in fermentation that ran for 54 hr sand 71 hrs, respectively. J2 medium, with lower concentrations of glucose and propionate at 20 g and 2 per liter, was supplemented with an extra dosage of tryptone at 4 g per liter. The fermentation with J2 medium was ran for 51 hrs. Extraction procedure employed for PHA recovery was refluxing with chloroform for 4 hrs, followed by precipitation in ethanol at various concentrations. The procedure was evaluated for molecular degradation using Fluka P(3HB) and P(3HB-co-3HV) (7:3) copolymer as reference materials. It was demonstrated that molecular degradation was low as 4 - 5% for P(3HB-co-3HV) precipitated in 75 - 85 % ethanol. For P(3HB), molecular degradation could not be evaluated as the Fluka reference material did not dissolve completely in the control solution. By combining the procedure of reflux and precipitation in 75% ethanol, purity of recovered PHA polymer could reach > 99% (referenced to Fluka P(3HB-co-3HV). However, there are bottlenecks to be eliminated from the procedure, such as filtration of biomass and handling of viscous PHA solution in bulk volume. The biomass harvested from J1 medium contained approximately 33% and 40% at 54 hrs and 71 hrs respectively, and 31% for J2 medium at 51 hrs. PHA polymer recovered from biomass samples were P(3HB-co-3HV) copolymer, as confirmed by the 'HNMR spectra, HB:HV was 82:18, 84:16 ad 80:20, respectively. The number average molecular weight (Mn) ranged from 1,240,000 to 1,420,000. With the incorporation of HV monomer units, melting temperature of the PHA polymer was lowered to 131 -137 C, giving a better thermal stability than the PHB homopolymer.

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